The aim of this study was to determine the quality of semen collected from stallions and chilled to +4 degrees C. Semen was collected from eight Polish cold-blooded stallions, aged 4-8 years. The ejaculate was examined macro- and microscopically to evaluate volume, concentration, viable sperm percentage, progressive motility, and morphology. The percentage of live/dead spermatozoa was determined by eosin and nigrosin staining. Flow cytometry was applied to detect phosphatidylserine translocation, whereas DNA defragmentation was determined with a commercial kit. The semen samples were examined immediately after collection and dilution (time 0) and at 6 storage times: 24, 48, 72, 96, and 120 hours later. Time-related changes in all the investigated spermatozoa parameters were observed at 6 different times. Progressive motility was negatively correlated with the percentage of live spermatozoa with phosphatidylserine translocation, but positively correlated with DNA defragmentation (P < 0.05). This study demonstrates that the evaluation of selected indicators of apoptosis is very useful in assessing the quality of chilled stallion semen. The results obtained show what significant changes occur in spermatozoa during storage at +4 degrees C. Evaluation of the cell membrane integrity and DNA defragmentation of spermatozoa makes it possible to determine whether and for how long semen chilled to +4 degrees C may be used for artificial insemination, which is extremely important during semen transportation. The methods of semen quality evaluation applied in this study should also be used in determining the usefulness of chilled semen for artificial insemination. This may lead to higher fertilization rates in mares
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